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Membrane vesicles (MVs) are spherical particles with nanoscale dimensions and characterized by the presence of diverse cargos, such as nucleic acids, proteins, lipids, and cellular metabolites. Many examples of (micro)organisms producing MVs are reported in literature. Among them, bacterial MVs are of particular interest because they are now considered as the fourth mechanism of horizontal gene transfer. Streptomyces bacteria are well-known for their ecological roles and ability to synthesize bioactive compounds, with Streptomyces coelicolor being the model organism. It was previously demonstrated that it can produce distinct populations of MVs characterized by different protein and metabolite cargos. In this work we demonstrated for the first time that MVs of S. coelicolor carry both DNA and RNA and that their DNA content represents the entire chromosome of the bacterium. These findings suggest that MV DNA could have a role in the evolution of Streptomyces genomes and that MVs could be exploited in new strain engineering strategies., (© 2022. The Author(s).)

Streptomyces coelicolor is a model organism for the study of Streptomyces , a genus of Gram-positive bacteria that undergoes a complex life cycle and produces a broad repertoire of bioactive metabolites and extracellular enzymes. This study investigated the production and characterization of membrane vesicles (MVs) in liquid cultures of S. coelicolor M145 from a structural and biochemical point of view; this was achieved by combining microscopic, physical and -omics analyses. Two main populations of MVs, with different sizes and cargos, were isolated and purified. S. coelicolor MV cargo was determined to be complex, containing different kinds of proteins and metabolites. In particular, a total of 166 proteins involved in cell metabolism/differentiation, molecular processing/transport, and stress response were identified in MVs, the latter functional class also being important for bacterial morpho-physiological differentiation. A subset of these proteins was protected from degradation following treatment of MVs with proteinase K, indicating their localization inside the vesicles. Moreover, S. coelicolor MVs contained an array of metabolites, such as antibiotics, vitamins, amino acids, and components of carbon metabolism. In conclusion, this analysis provides detailed information on S. coelicolor MVs under basal conditions and on their corresponding content, which may be useful in the near future to elucidate vesicle biogenesis and functions. IMPORTANCE Streptomycetes are widely distributed in nature and characterized by a complex life cycle that involves morphological differentiation. They are very relevant in industry because they produce about half of all clinically used antibiotics, as well as other important pharmaceutical products of natural origin. Streptomyces coelicolor is a model organism for the study of bacterial differentiation and bioactive molecule production. S. coelicolor produces extracellular vesicles that carry many molecules, such as proteins and metabolites, including antibiotics. The elucidation of S. coelicolor extracellular vesicle cargo will help us to understand different aspects of streptomycete physiology, such as cell communication during differentiation and response to environmental stimuli. Moreover, the capability of these vesicles for carrying different kinds of biomolecules opens up new biotechnological possibilities related to drug delivery. Indeed, decoding the molecular mechanisms involved in cargo selection may lead to the customization of extracellular vesicle content.

The actinomycetes, Gram-positive filamentous bacteria, are the most prolific source of natural occurring antibiotics. At an industrial level, antibiotics from actinomycete strains are produced by means of submerged fermentations, where one of the major factors negatively affecting bioproductivity is the pellet-shaped biomass growth. The immobilization of microorganisms on properly chosen supports prevents cell-cell aggregation resulting in improving the biosynthetic capability. Thus, novel porous biopolymer-based devices are developed by combining melt mixing and particulate leaching. In particular, polycaprolactone (PCL), polyethylene glycol (PEG), and sodium chloride (NaCl) with different grain sizes are used to prepare PCL/PEG/NaCl blends in the melt. These blends are then leached to obtain PCL-based porous membranes that are used as solid support for the growth of Streptomyces coelicolor, a model streptomycete used to produce various antibiotics including the blue colored actinorhodin (ACT). Thereafter, the effect of the devices' characteristics on the bacterial growth and on the production ACT is evaluated. The results showed that ACT production is strongly dependent on the pore size distribution of the device. Moreover, membranes with pores ranging from 90 to 110 μm are able to offer a potential improvement in volumetric productivity of ACT if compared to conventional submerged liquid culture., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The molecular mechanisms regulating tryptophan biosynthesis in actinomycetes are poorly understood; similarly, the possible roles of tryptophan in the differentiation program of microorganism life-cycle are still underexplored. To unveil the possible regulatory effect of this amino acid on gene expression, an integrated study based on quantitative teverse transcription-PCR (qRT-PCR) and proteomic approaches was performed on the actinomycete model Streptomyces coelicolor. Comparative analyses on the microorganism growth in a minimal medium with or without tryptophan supplementation showed that biosynthetic trp gene expression in S. coelicolor is not subjected to a negative regulation by the presence of the end product. Conversely, tryptophan specifically induces the transcription of trp genes present in the biosynthetic gene cluster of the calcium-dependent antibiotic (CDA), a lipopeptide containing D- and L-tryptophan residues. In addition, tryptophan stimulates the transcription of the CDA gene cluster regulator cdaR and, coherently, CDA production. Surprisingly, tryptophan also promotes the production of actinorhodin, another antibiotic that does not contain this amino acid in its structure. Combined 2D-DIGE and nano liquid chromatography electrospray linear ion trap tandem mass spectrometry (LC-ESI-LIT-MS/MS) analyses revealed that tryptophan exerts a growth-stage-dependent global effect on S. coelicolor proteome, stimulating anabolic pathways and promoting the accumulation of key factors associated with morphological and physiological differentiation at the late growth stages. Phenotypic observations by scanning electron microscopy and spore production assays demonstrated an increased sporulation in the presence of tryptophan. Transcriptional analysis of catabolic genes kynA and kynB suggested that the actinomycete also uses tryptophan as a carbon and nitrogen source. In conclusion, this study originally provides the molecular basis underlying the stimulatory effect of tryptophan on the production of antibiotics and morphological development program of this actinomycete.

The alkB gene, encoding an alkane monooxygenase in the actinomycete Gordonia sp. SoCg, was expressed in the non-alkane-degrading actinomycete Streptomyces coelicolor M145. The resulting engineered strain, M145-AH, can grow on n-hexadecane as sole carbon source. To unravel proteins associated with growth on n-alkanes, proteome of M145-AH after 6, 24, and 48 h of incubation in the Bushnell-Haas (BH) mineral medium containing n-hexadecane as sole carbon source (H condition) and in BH without any carbon source (0 condition) were compared using 2D-differential gel electrophoresis. Proteome analysis revealed significant changes only at 48 h, showing 48 differentially abundant proteins identified by mass spectrometry procedures. To asses if these proteins were specifically related to n-hexadecane metabolism, their expression was investigated, comparing H proteome with that of M145-AH incubated in BH with glucose as sole carbon source (G condition). Thus, protein expression profiles at 6, 24, and 48 h under H, 0, and G conditions were combined, revealing that M145-AH regulates in a temporally- and carbon source-dependent manner the expression of proteins involved in regulatory events, central carbon metabolism, respiration, β-oxidation, membrane transport, and amino acid and protein metabolism. Interestingly, 21 % of them, mostly involved in membrane transport and protein metabolism, showed a n-hexadecane-dependent regulation with regulatory proteins such as CRP likely to have a key role in M145-AH n-hexadecane growth. These results, expanding the knowledge on n-alkane utilization in Gram-positive bacteria, reveal genes to be targeted to develop an efficient S. coelicolor M145-AH-based bioremediation system.

Most enzymes involved in tryptophan catabolism via kynurenine formation are highly conserved in Prokaryotes and Eukaryotes. In humans, alterations of this pathway have been related to different pathologies mainly involving the central nervous system. In Bacteria, tryptophan and some of its derivates are important antibiotic precursors. Tryptophan degradation via kynurenine formation involves two different pathways: the eukaryotic kynurenine pathway, also recently found in some bacteria, and the tryptophan-to-anthranilate pathway, which is widespread in microorganisms. The latter produces anthranilate using three enzymes also involved in the kynurenine pathway: tryptophan 2,3-dioxygenase (TDO), kynureninase (KYN), and kynurenine formamidase (KFA). In Streptomyces coelicolor, where it had not been demonstrated which genes code for these enzymes, tryptophan seems to be important for the calcium- dependent antibiotic (CDA) production. In this study, we describe three adjacent genes of S. coelicolor (SCO3644, SCO3645, and SCO3646), demonstrating their involvement in the tryptophan-to-anthranilate pathway: SCO3644 codes for a KFA, SCO3645 for a KYN and SCO3646 for a TDO. Therefore, these genes can be considered as homologous respectively to kynB, kynU, and kynA of other microorganisms and belong to a constitutive catabolic pathway in S. coelicolor, which expression increases during the stationary phase of a culture grown in the presence of tryptophan. Moreover, the S. coelicolor ΔkynU strain, in which SCO3645 gene is deleted, produces higher amounts of CDA compared to the wild-type strain. Overall, these results describe a pathway, which is used by S. coelicolor to catabolize tryptophan and that could be inactivated to increase antibiotic production.

Through the screening of a Streptomyces coelicolor genomic library, carried out in a histidinol phosphate phosphatase (HolPase) deficient strain, SCO5208 was identified as the last unknown gene involved in histidine biosynthesis. SCO5208 is a phosphatase, and it can restore the growth in minimal medium in this HolPase deficient strain when cloned in a high or low copy number vector. Moreover, it shares sequence homology with other HolPases recently identified in Actinobacteria. During this work a second phosphatase, SCO2771, sharing no homologies with SCO5208 and all so far described phosphatases was identified. It can complement HolPase activity mutation only at high copy number. Sequence analysis of SCO5208 and SCO2771, amplified from the HolPase mutant strain, revealed that SCO5208 shows a mutation in a conserved amino acid, whereas SCO2771 does not show any mutation. All these results show that S. coelicolor SCO5208, recently renamed hisN, is the HolPase involved in histidine biosynthesis.

HspR (heat shock protein regulator) acts as a negative regulator of different genes in many bacteria. In Streptomyces coelicolor hspR gene is part and the transcriptional repressor of the dnaK operon which encodes the DnaK, GrpE, DnaJ chaperone machines and HspR itself. Our experiments led us to the discovery of a second promoter, internal to dnaK operon, located upstream hspR gene. Transcription from this promoter was detected at 30 degrees C indicating that hspR could play a key physiological role.

Aims: Characterization of SCP2165, a plasmid identified in the Gram-positive bacterium Streptomyces coelicolor A3(2)., Methods and Results: Pulsed-field gel electrophoresis (PFGE) of mycelia of a S. coelicolor strain embedded in low melting agarose revealed the presence of a plasmid. Restriction enzyme mapping and sequence analysis of a 2.1 kb fragment revealed that this plasmid could be SCP2. SCP2 and its spontaneous derivative SCP2* are self-transmissible plasmids and have chromosome mobilizing ability (c.m.a.). SCP2* has a c. 1000-fold increased c.m.a. compared with SCP2. Interestingly the plasmid, named SCP2165, shows a c.m.a. from 5x10(-2) to 1x10(-1) which is 50-100-fold higher than that described for crosses involving SCP2*., Conclusions: SCP2165 is a SCP2 derivative plasmid with the highest c.m.a. so far described for SCP2 derivative plasmids. PFGE, under conditions we used, seems to be a fast way to identify large circular plasmids in Streptomyces strains., Significance and Impact of the Study: Further knowledge of the SCP2 family may allow the construction of improved SCP2-derived cloning vectors. SCP2165 could be a potential tool for conjugational transfer of gene clusters between different Streptomyces species.